The research proposed here is directed toward an understanding of the molecular mechanism of general genetic recombination. This goal is approached through the study of special sites in DNA promoting a high rate of recombination in their vicinity and through the identification and study of the activity of proteins interacting with these sites. The Chi recombinational hotspots of bacteriophage lambda and its host Escherichia coli, and the M26 recombinational hotspot of the fission yeast Schizosaccharomyces pombe will be studied in the proposed research. In addition, the interaction of Chi and RecBCD enzyme, which directly activates Chi, site will be sought. These studies will elucidated the molecular mechanisms by which recombinational hotspots act and , hence, elucidate an important (rate-limiting) step in homologous recombination. Specific aims are to: (1) Determine if RecBCD enzyme is altered after encountering Chi. (2) Couple the production of ssDNA ends at Chi by RecBCD enzyme to the production of hybrid DNA by RecA and SSB proteins. (3) Locate with high resolution the point of Chi-stimulated genetic exchange, and determine whether the action of cellular enzymes, such as nucleases, alters this point. (4) Determine whether Chi acts at an early or late step of recombination. (5) Search for Chi-dependent DNA strand cleavage by RecBCD enzyme in E. coli cells. (6) Determine the basis of the context- dependence of the M26 hotspot activity in S. pombe. (7) Isolate mutants of S. pombe that fail to activate the M26 hotspot, or that activate other nucleotide sequences as hotspots. (8) Analyze S. pombe rec mutants for locus specificity. Recombination plays an important role in generating diversity among individuals in a population and, in some cases, among cells within an organism. Understanding the mechanism of recombination thus lends insight into the evolution and development of organisms. Aberrations of recombination may be responsible for certain chromosomal rearrangements associated with birth defects and cancer.